LPAAT-beta inhibitors and uses thereof

ABSTRACT

The invention relates to triazines and the use thereof to inhibit lysophosphatidic acid acyltransferase β (LPAAT-β) activity. The invention further relates to methods of treating cancer using said triazines. The invention also relates to methods for screening for LPAAT-β activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of allowed U.S. patent applicationSer. No. 11/224,340 filed Sep. 12, 2005; which application is adivisional of U.S. patent application Ser. No. 10/712,900 filed Nov. 13,2003, which issued as U.S. Pat. No. 7,064,125; which application is adivisional of U.S. patent application Ser. No. 10/236,084 filed Sep. 06,2002, abandoned; which application is a continuation of U.S. patentapplication Ser. No. 09/984,888 filed Oct. 31, 2001, abandoned, whichclaims priority to U.S. Provisional Application Ser. No. 60/244,195,filed Oct. 31, 2000, abandoned, the disclosures of which areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is in the field of organic and medicinal chemistry. Inparticular, the invention relates to triazines and the use thereof toinhibit lysophosphatidic acid acyltransferase β (LPAAT-β) activity. Theinvention further relates to methods of treating cancer using saidtriazines. The invention also relates to methods for screening forLPAAT-β activity.

2. Related Art

LPAAT catalyzes the acylation of lysophosphatidic acid (LPA) tophosphatidic acid. LPA is the simplest glycerophospholipid, consistingof a glycerol molecule, a phosphate group, and a fatty acyl chain. LPAATadds a second fatty acyl chain to LPA, producing phosphatidic acid (PA).PA is the precursor molecule for certain phosphoglycerides, such asphosphatidylinositol, and diacylglycerols, which are necessary for theproduction of other phosphoglycerides, such as phosphatidylcholine, andfor triacylglycerols, which are essential biological fuel molecules.

In addition to being a crucial precursor molecule in biosyntheticreactions, LPA has recently been added to the list of intercellularlipid messenger molecules. LPA interacts with G protein-coupledreceptors, coupling to various independent effector pathways includinginhibition of adenylate cyclase, stimulation of phospholipase C,activation of MAP kinases, and activation of the small GTP-bindingproteins Ras and Rho. Moolenaar, J. Biol. Chem 28:1294 (1995). Thephysiological effects of LPA have not been fully characterized as yet.However, one of the physiological effects that is known is that LPApromotes the growth and invasion of tumor cells. It has been shown thatthe addition of LPA to ovarian or breast cancer cell lines induces cellproliferation, increases intracellular calcium levels, and activates MAPkinase. Xu et al., Biochem. J. 309:933 (1995). In addition, LPA has beenshown to induce MM1 tumor cells to invade cultured mesothelial cellmonolayers. Imamura et al. Biochem. Biophys. Res. Comm. 193:497 (1993).

Like LPA, PA is also a messenger molecule. PA is a key messenger in acommon signaling pathway activated by proinflammatory mediators such asinterleukin-1β, tumor necrosis factor α, platelet activating factor, andlipid A. Bursten et al., Am. J. Physiol. 262:C328 (1992); Bursten etal., J. Biol. Chem. 255:20732 (1991); Kester J. Cell Physiol. 156:317(1993). PA has been implicated in mitogenesis of several cell lines[English, Cell Signal 8: 341 (1996)]. PA level has been found to beincreased in either ras or fps transformed cell lines compared to theparental Rat2 fibroblast cell line [Martin et al., Oncogene 14: 1571(1997)]. Activation of Raf-1, an essential component of the MAPKsignaling cascade, by extracellular signals is initiated by associationwith intracellular membranes. Recruitment of Raf-1 to membranes has beenreported to be mediated by direct association with phosphatidic acid[Rizzo et al., J. Biol Chem 275:23911-8 (2000)]. Thus, LPAAT, as anenzyme that regulate PA content in cells, may play a role in cancer, andmay also mediate inflammatory responses to various proinflammatoryagents.

SUMMARY OF THE INVENTION

The preferred embodiments of the present invention relate to a compoundof the Formula:

wherein,

R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

R², R³, R⁴ and R⁵, each of which may be same or different, are hydrogen,alkyl, substituted alkyl, alkenyl, alkynyl, aryl or substituted aryl; or

R² and R³ or R⁴ and R⁵, together with the nitrogen to which they areattached, form a piperidine, piperazine, or a morpholine ring; or

pharmaceutically acceptable salts thereof.

The preferred embodiments of the present invention further relate to amethod for inhibiting LPAAT-β (lysophosphatidic acid acyltransferase β)comprising contacting LPAAT-β with an effective amount of a compound ofthe Formula:

wherein,

R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

R², R³, R⁴ and R⁵, each of which may be same or different, are hydrogen,alkyl, substituted alkyl, alkenyl, alkynyl, aryl or substituted aryl; or

R² and R³ or R⁴ and R⁵, together with the nitrogen to which they areattached, form a piperidine, piperazine, or a morpholine ring; or

pharmaceutically acceptable salts thereof;

thereby inhibiting LPAAT-β.

The preferred embodiments of the present invention further relate to amethod of inhibiting cell proliferation comprising contacting a cellwith an effective amount of a compound of the Formula:

wherein,

R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, arylox orsubstituted amino;

R², R³, R⁴ and R⁵, each of which may be same or different, are hydrogen,alkyl, substituted alkyl, alkenyl, alkynyl, aryl or substituted aryl; or

R² and R³ or R⁴ and R⁵, together with the nitrogen to which they areattached, form a piperidine, piperazine, or a morpholine ring; or

pharmaceutically acceptable salts thereof;

thereby inhibiting the proliferation of the cell.

The preferred embodiments of the present invention further relate to amethod for treating cancer, comprising administering to an animal inneed thereof, an effective amount of a compound of the Formula:

wherein,

R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

R², R³, R⁴ and R⁵, each of which may be same or different, are hydrogen,alkyl, substituted alkyl, alkenyl, alkynyl, aryl or substituted aryl; or

R² and R³ or R⁴ and R⁵, together with the nitrogen to which they areattached, form a piperidine, piperazine, or a morpholine ring; or

pharmaceutically acceptable salts thereof;

wherein the cancer is treated.

The preferred embodiments of the present invention further relate to amethod for screening a patient for LPAAT-β activity, said methodcomprising detecting the presence or absence of an increased amount ofLPAAT-β RNA, DNA or protein relative to a predetermined control, wherebythe presence of said increased amount is indicative of cancersusceptibility in said patient.

The preferred embodiments of the present invention further relate to amethod of inhibiting cell proliferation comprising the inhibition ofLPAAT-β.

The preferred embodiments of the present invention further relate to avaccine preparation capable of inducing an anti-tumor immune responsecomprising a pharmaceutically acceptable carrier and an anti-tumorimmune response-inducing effective amount of LPAAT-β protein.

The preferred embodiments of the present invention further relate to amethod for screening a patient for LPAAT-β activity, said methodcomprising detecting the presence or absence of an increased amount of aphospholipid of defined acyl-chain composition relative to apredetermined control, whereby the presence of said increased amount isindicative of cancer susceptibility in said patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results on a breast intraductal adenocarcinoma samplewhere there is moderate increase in LAPPT-β mRNA level in the tumorsample (top two panels).

FIG. 2 shows the results on a breast intraductal adenocarcinoma samplewhere there is a large increase in LPAAT-β mRNA level in the tumorsample.

FIG. 3 shows three examples of ovarian cancer where the LPAAT-β mRNAlevels are elevated and one example with undetectable level of LPAAT-βmRNA (lower right panel).

FIG. 4A shows the results on a prostate adenocarcinoma sample wherethere is moderate increase in LPAAT-β mRNA level in the tumor sample.

FIG. 4B shows the results on immunohistochemical staining of ovariantissue with MoAb 4B12.

FIG. 4C shows the results on immunohistochemical staining of cervicaltissue with MoAb 4B12.

FIG. 4D shows the results on immunohistochemical staining of lung tissuewith MoAb 4B12.

FIG. 4E shows the summary of immunohistochemistry results of the varioustissue samples stained by MoAb 4B12.

FIG. 5A shows hemacytometer cell counts of ECV304 cell lines.

FIG. 5B shows examples of cell morphology of NIH/3T3 cells afterexposure to specified agents.

FIG. 5C shows the growth profiles of transduced populations of NIH/3T3cells.

FIG. 5D shows the growth profiles of transduced populations of LNCaPcells.

FIG. 5E shows the effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on the proliferationof MCF-7 cells.

FIG. 6A shows detection of tumor formation from LPAAT-β overexpressingcells.

FIG. 6B shows the effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on the volume oftumors in mice.

FIG. 6C shows the effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on the growth of B16melanoma cells.

FIG. 6D shows the effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on the growth ofLewis Lung tumor cells.

FIG. 6E shows the effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on the growth ofDU145 prostate tumor cells.

FIG. 7 shows a colorimetric assay whose time course of color developmentis dependent on LPAAT enzyme.

FIG. 8 shows the result from assaying a plate of various compounds at 16mM.

FIG. 9 shows the results of the effects of a compound selected fromsecondary screening on LPAAT-β activity and LPAAT-A activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. LPAAT-α and LPAAT-β: An Overview.

Northern blot analysis shows that LPAAT-α is expressed in all humantissues tested with the highest expression level found in skeletalmuscle (West et al. DNA Cell Biol. 16:691 (1997)). The uniformity ofLPAAT-α expression has also been found in additional tissues such asprostate, testis, ovary, small intestine, and colon (Stamps et al.,Biochem. J. 326: 455 (1997)) as well as in mouse tissues (Kume et al.,Biochem. Biophys. Res. Commun. 237: 663 (1997)). A 2 kb and a 1.3 kbforms, possibly due to alternative utilization of polyadenylationsignals at the 3′-UTR, have been found in murine LPAAT-α mRNA (Kume etal., Biochem. Biophys. Res. Commun 237: 663 (1997)), whereas only onemajor human LPAAT-α mRNA of 2 kb in size has been detected by Northernanalysis. West et al., DNA Cell Biol. 16: 691 (1997); Stamps et al.,Biochem. J. 326: 455 (1997).

In contrast, LPAAT-β demonstrates a distinct tissue distribution of mRNAexpression. West et al., DNA Cell Biol. 16: 691 (1997). LPAAT-β is mosthighly expressed in liver and heart tissues. LPAAT-β is also expressedat moderate levels in pancreas, lung, skeletal muscle, kidney, spleen,and bone marrow; and at low levels in thymus, brain and placenta. Thisdifferential pattern of LPAAT-β expression has been confirmedindependently (Eberhardt et al., J. Biol. Chem. 272: 20299 (1997)) withthe only discrepancy being that high level, instead of moderate level,of LPAAT-β has been detected in pancreas, possibly due to slight lotvariations in commercial RNA blots (Clontech, Palo Alto, Calif.). Inaddition, moderate LPAAT-β expression has been found in prostate,testis, ovary, small intestine, and colon with the small intestinecontaining relatively higher amounts. Eberhardt et al., J. Biol Chem272: 20299 (1997). Within various brain sections, high expression hasbeen found in the subthalamic nucleus and spinal cord; and least in thecerebellum, caudate nucleus, corpus callosum, and hippocampus. LPAAT-βcan also be detected in myeloid cell lines THP-1, HL-60, and U937 withthe mRNA levels remaining the same with or without phorbal-estertreatment. The size difference between human LPAAT-α and LPAAT-β mRNA isconsistent with the sequence data, in which LPAAT-α has a longer 3′-UTR.The differential tissue expression pattern LPAAT-α and LPAAT-β mRNAwould suggest these two genes are regulated differently and are likelyto have independent functions. Therefore, a desirable feature incompounds that inhibit LPAAT activity is that they are specific ininhibiting one isoform of the enzyme over the other (i.e., LPAAT-β overLPAAT-α).

II. LPAAT-β and Cancer.

PA has been implicated in mitogenesis of several cell lines. English,Cell Signal 8: 341 (1996). PA level has been found to be increased ineither ras or fps transformed cell lines compared to the parental Rat2fibroblast cell line (Martin et al., Oncogene 14: 1571 (1997). To testwhether LPAAT expression may be enhanced in certain tumor cells, theexpression of LPAAT-α and LPAAT-β mRNA in human tumor panel blots(Invitrogen, Carlsbab, Calif.) that contained tumor RNAs, isolated fromvarious malignant tissues and RNAs from the normal tissues in thesurgical margins, were examined. Leung et al., DNA Cell Biol. 17: 377(1998). The same blots were also reprobed using cDNAs encodingphosphatidic acid phosphatase isoform PAP2-α; an enzyme that degrades,rather than generates, PA. Of a total of eight different tissuesexamined, LPAAT-β mRNA was found to be elevated in three tumors tissues(uterus, fallopian tube, and ovary), as compared to its expression inthe corresponding normal tissues. However, no significant difference wasfound in LPAAT-α mRNA level between the various tumor tissues and thenormal adjacent tissues. In two of the tumor tissues (fallopian tube andovary) where LPAAT-α mRNA was elevated, PAP2-α mRNA expression was foundto be suppressed, as it was also in tumors of the colon, rectum, andbreast.

Since the finding of differential expression of LPAAT-β mRNA in certaintumor versus normal tissues is based on Northern analysis of a singlespecimen from a given tissue, more studies will be needed to determinewhether the relative elevation of LPAAT-β expression in selected tumortissues can be applied and extended to similar tissues derived from alarger number of donors. Leung et al., DNA Cell Biol. 17: 377 (1998).Accordingly, in situ hybridization was used to compare LPAAT-β mRNAlevels in breast, ovary, and prostate tumor samples obtained frommultiple independent donors (LifeSpan Biosciences, Seattle, Wash.).Specifically, the coding region of human LPAAT-β was amplified by PCRfrom the plasmid pCE9. LPAAT-β with primers 5′-GCATGAATTC AAAGGCCTACGTCGACATGG AGCTGTGGCC GTG-3′ (SEQ ID NO:1) and 5′-GTCGACTCTA GACTACTGGGCCGGCTGCAC-3′ (SEQ ID NO:2). The resultant 870 bp PCR product was thencut with EcoR I and XbaI for insertion in between the EcoR I and XbaIsites of the in vitro transcription vector pDP18-T7/T3 (Ambion, Austin,Tex.) to generate the plasmid pDP_(—)1ptB. Serial tissue sections fromparaffin archival samples were hybridized with digoxigenin labeledriboprobes transcribed from either a T3 (sense) or T7 (antisense)transcription initiation site present in the plasmid pDP_(—)1ptBlinearized with either EcoR I (antisense) or Xba I (sense). The tissuesections from paraffin blocks were digested with proteinase K (20 μg/ml)for 4 minutes, then hybridized with the antisense probe (1 μg/ml) at 60°C. for 22 hours and subsequently washed with 2×SSC and 0.1×SSC at 50° C.The hybridization signals were detected with NBT/BCIP substrates usingthree cycles of an alkaline phosphatase TSA amplification system (NENLife Sciences, Boston, Mass.). The specimens were then counterstainedwith methyl green. The signal was developed within 30 minutes at roomtemperature. The slides were then imaged using a digital camera mountedonto a microscope.

Breast and ovary tissues were chosen for further in situ hybridizationstudy, as initial Northern analysis showed elevation of LPAAT-β mRNAlevels in tumors derived from the female reproductive tract. Prostatetissue was chosen, as it responds to steroid hormones and containsductal structures in a manner similar to breast and ovary tissues. Usingan anti-sense cDNA probe, it was demonstrated that expression of the βisoform of this enzyme (LPAAT-β) was augmented in human tumor tissue in10/11 ovarian, 14/20 breast, and 7/16 prostate biopsies as compared tonormal adjacent tissues. FIG. 1 shows an example of the results on abreast intraductal adenocarcinoma sample where there is moderateincrease in LPAAT-β mRNA level in the tumor samples (top 2 panels) asevidenced by more dark-purple to brown spots compared to adjacenthyperplasia (bottom-left panel) and normal tissue (bottom-right panel).The slight increase in LPAAT-β mRNA staining in the hyperplasia sample(bottom-left panel) versus the normal sample (bottom-right panel)suggests that elevation occurs at an early stage of oncogenesis. FIG. 2shows an example of the results on another breast intraductaladenocarcinoma sample where there is large increase in LPAAT-β mRNAlevel in the tumor sample (left panel) as evidenced by more dark-purplespots versus the adjacent normal tissue (right panel). FIG. 3 showsthree examples of ovarian cancer samples where the LPAAT-β mRNA levelsare elevated and one example with undetectable level of LPAAT-β mRNA(lower right panel). FIG. 4A shows an example of the results on aprostate adenocarcinoma sample where there is moderate increase inLPAAT-β MRNA level in the tumor samples (left panel) as evidenced bymore dark-purple spots versus the adjacent normal tissue (right panel).In no cases have elevated levels of LPAAT-β mRNA expression been foundin the adjacent normal region from the same donor even in those cases ofbreast, ovarian, or prostate tumor where LPAAT-β mRNA levels happen tobe low or undetectable. The augmented expression of LPAAT-β in a highpercentage of tumor samples from breast (70%), ovary (91%), and prostatetissues (44%) would suggest that LPAAT-β overexpression may be acontributing factor for the development of these tumors.

To determine if increased transcription of LPAAT-β mRNA in selectedtumor tissues can be extended to increased LPAAT-β protein expression ina wider range of tissues, a monoclonal antibody specific for humanLPAAT-β protein (MoAb 4B12) was generated based on the petide sequence,DLGERMVRENLKVW, derived from amino acids 155-168 of LPAAT-β protein(BAbCO, Berkeley, Calif.). FIG. 4B shows an example of the results onimmunohistochemical staining (PhenoPath, Seattle, Wash.) with MoAb 4B12at 1:4000 dilution of ovarian tissue where there is substantial increasein LPAAT-β protein expression in the tumor samples (right panels) asevidenced by more intense brown stainings versus the normal tissue (leftpanel). FIG. 4C shows an example of the results on immunohistochemicalstaining (PhenoPath, Seattle, Wash.) with MoAb 4B12 at 1:4000 dilutionof cervical tissue where there is substantial increase in LPAAT-βprotein expression in the tumor samples (right panels) as evidenced bymore intense brown stainings versus the normal tissue (left panel).There is also more staining in the surrounding stromal cells (indicatedby arrows) in the tumor tissue vs the normal tissue, suggesting that thetumor may also induce LPAAT-β protein expression in the surroundingcells. FIG. 4D shows another example of the results onimmunohistochemical staining (PhenoPath, Seattle, Wash.) with MoAb 4B12at 1:4000 dilution of lung tissue where there is extensive increase inLPAAT-β protein expression in the tumor samples (right panels) asevidenced by more intense brown stainings versus the normal tissue (leftpanel). FIG. 4E shows the summary of immnohistochemistry (IHC) resultsof the various tissue samples stained by MoAb 4B12. The augmentedexpression of LPAAT-β in a high percentage of tumor samples againsuggest that LPAAT-β overexpression may be a contributing factor for thedevelopment of these tumors and that LPAAT-β may be a useful target forthe development of anti-cancer compounds.

The aforementioned antibody may also be used for diagnostic andprognostic purposes when a tumor is present both on biopsies and inserum or plasma. For example, ELISA may be performed on serum to detectlung or ovarian cancer. It should be mentioned that currently there areno useful early diagnostics for these types of cancers.

The overexpression of LPAAT-β in selected tumor tissues would alsosuggest the LPAAT-β protein may constitute a useful antigen for thedevelopment of tumor vaccines against those tumors where LPAAT-β isoverexpressed. Fong et al., Annu. Rev. Immunol. 18: 245 (2000);Schreurs, et al., Crit. Rev. Oncol. 11: 1 (2000). One such approach mayuse autologous dendritic cells, a type of potent antigen-presentingcells, to present LPAAT-β as a tumor-associated antigens for thegeneration of tumor-specific immunity through the MHC class I and IIprocessing pathways. Administration of dendritic cells loaded ex vivowith LPAAT-β as a therapeutic vaccine to patients with tumors withaugmented LPAAT-β expression may induce T cell-mediated tumordestruction.

To assess whether LPAAT-β overexpression in cells would lead to certainphenotypic changes that are commonly observed in transformed cells, thegrowth and adherence characteristics of ECV304 cells (American TypeCulture Collection, Richmond, Va.) expressing LPAAT-β (LPTb), expressinga catalytically inactive form of LPAAT-β (b-M8) whereby the arginine atposition 175 was changed to alanine using the GeneEditor™ in vitrosite-directed mutagenesis system (Promega, Madison, Wis.), or expressinggreen fluorescent protein (GFP) as a control were compared. Theaforementioned cells that express GFP may be considered to be anon-limiting example of a “predetermined control,” according to thepreferred embodiments of the present invention. That is, such cells maybe used to gauge whether a cell is over- or under-expressing LPAAT-βDNA, RNA or protein. FIG. 5A shows the growth curve of these three celllines. Each cell line was seeded at 200,000 cells per 60 mm plate. Thecell numbers at various times after seeding were determined by countingwith a hemacytometer. The growth rate of the three cell lines weresimilar until they reached confluence at 100 hours after plating. Afterconfluence, the LPTb cells were able to continue to proliferate, whilethe b-M8 and GFP cells' growth started to level off. This demonstratedthat ECV304 cells overexpressing LPAAT-β could continue to grow andcould form a plurality of layers after they had formed a confluentmonolayer of cells. The proliferation of the cells with the inactivemutant or the control cells slowed down after confluence. The loss ofcontact inhibition and the propensity for growth to an unusually highcell density are changes commonly observed in tumorigenesis. The factthat the inactive LPAAT-β mutant (b-M8) expressing cells, like thevector control cells, are constrained by density-dependent inhibition ofcell division strongly suggests that the capacity to overcome contactinhibition may be due to increases in LPAAT-β enzymatic activity. Thedevelopment of compounds that inhibit LPAAT-β enzymatic activity mayreverse the growth pattern and hence tumorigenesis in cells withabnormally high level of LPAAT-β expression.

To determine if the observation from LPAAT-β expressing ECV304 cells canbe extended to other cell types and to animal models of tumorigenesis,LPAAT-β cDNA was inserted into a retroviral expression vector, pLOXSN,for the generation of recombinant viral stocks in a packaging cell line,PT67 (Clontech, Palo Alto, Calif.), for transduction into various celllines. The vector pLOXSN was derived from pLXSN with insertion of a 19bp oligonucleotide coding for the locus of recombination (lox) signalsequence as well as a Clal recognition site into the NheI site withinthe 3′-LTR region of pLXSN. Miller and Rosman BioTechniques 7: 980(1989); Hoess. and Abremski, Nucleic Acid and Mol. Biol. 4: 99 (1990).This lox sequence will be duplicated within the 5′-LTR region duringviral replication. Hence the sequence in between the two lox siteslocated within the 5′- and the 3′-LTR can be excised if required in thepresence of the enzyme cre recombinase supplied in trans from a separateretroviral vector with a different selectable marker.

Over-expression of the normal cellular LPAAT-β cDNA in NIH/3T3 cells wasassociated with transformation in 3 out of 9 transduced populations. Asis the case with normal cellular proto-oncogenes, over-expression ofLPAAT-β is not sufficient, but may contribute to transformation alongwith other, spontaneous events. FIG. 5B shows examples of cellmorphology of NIH/3T3 cells: a bulk population transfected with aplasmid overexpressing the Ki-ras oncogene (top left panel), a selectedclone transduced with a retroviral vector overexpressing LPAAT-β (Hc2;lower left panel) and cells with the LPAAT-β cDNA excised using thelox-cre recombination in the lower left and normal, untransduced cells(top right panel). Sauer, Methods 14: 381 (1998). The controluntransduced cells exhibited normal fibroblast morphology and grew as acontact-inhibited, adherent monolayer (top right panel). In contrast,both the Ki-ras and LPAAT-β overexpressing cells were more elongated andspiked, were not contact-inhibited and formed foci typical oftransformation of these immortalized fibroblasts. After removal LPAAT-βtransgene by lox-cre recombination from the Hc2 clone (bottom rightpanel), this transformed morphology was lost, suggesting that LPAAT-βoverexpression is a contributing factor to this transformation phenotyperather than being the result entirely of spontaneous events during invitro passage.

Another common parameter of cancer cells is a reduced requirement forelements present in serum. FIG. 5C compares the growth profiles oftransduced populations of NIH/3T3 cells in low (2%) serum. Twoindependent populations (LPT Hc2, LPT L bulk) overexpressing LPAAT-βhave an increased ability to proliferate compared to a control vectorclone expressing alkaline phosphatase (APc1) and those correspondingpopulations with deletion of the LPAAT-β transgene by lox-crerecombination (LPT Hc2cre, LPT L bulkcre), suggesting that LPAAT-βoverexpression is a contributing factor to this transformed phenotype ofproliferation with a reduced requirement for growth factors.

Similarly, out of a total of 12 populations of human prostate LNCaPcells (American Type Culture Collection, Manassas, Va.) transduced withLPAAT-β expressing vector, most of them show augmented proliferation inlow serum when compared to control cells (FIG. 5D).

To determine whether administration of LPAAT-β inhibitor would have anyeffect on cell proliferation in tissue culture, proliferation of humanbreast tumor MCF-7 cells in microplates were measured by CyQUANTanalysis using a green-fluorescent nucleic acid stain optimized toproduce a linear detection range from 50 to 50,000 cells in 200 μlvolume (Molecular Probes, Eugene, Oreg.) in the presence of variousconcentrations of a LPAAT-β inhibitor. FIG. 5E shows the triazinecompound shows 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine at >20μM is effective in blocking the proliferation of MCF-7 cells.

To determine if LPAAT-β overexpression would contribute to tumorigenesisin mice, 2×10⁶ NIH3T3 cells overexpressing LPAAT-β (LPAAT vector) andcontrol cells were injected subcutaneously into nude mice. FIG. 6A showstumor could be detected after 14 days from the LPAAT-p overexpressingcells, while no tumor formation was detected in vector control cellsafter 28 days. The cells with the transgene removed by lox-crerecombination showed delay of tumor formation compared to LPAAT-βoverexpressing cells by ˜7 days. Recovery and analysis of the lox-crecells from mice showed that there had been in vivo selection of a smallsub-population that had not been recombined to remove the LPAAT-βtransgene. This analysis demonstrated that the only cells to form tumorsretained the original LPAAT vector and indeed had a high level of LPAATactivity as well as G418 resistance (the neo gene is also removed alongwith LPAAT-β during the cre-lox procedure). These data show LPAAT-βoverexpression is a contributing factor for tumorigenesis in vivo.

To determine whether administration of LPAAT-β inhibitor would have anyeffect on tumor growth in mice, 5×10⁵ NIH/3T3 cells overexpressing theoncogene Ki-ras were injected subcutaneously into nude mice. An LPAAT-βinhibitor 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine, atconcentrations that range from 10 mg/Kg to 100 mg/Kg of mouse bodyweight was injected intra-peritoneally on day 1, 2, 3 and 4 afterinjection of tumor cells. The size of tumors was then measured on day 8.FIG. 6B shows the volume of the tumors in mice is decreased as theconcentration of the LPAAT-β inhibitor increases, suggesting thatadministration of this LPAAT-β inhibitor is efficacious in slowing downtumor growth in vivo.

In addition to slowing down tumor growth of NIH3T3/Ki-ras cells in nudemice, 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine has also beenfound to decrease the growth of B16 melanoma cells (FIG. 6C) and LewisLung tumor cells (FIG. 6D) in syngeneic mice as well as the growth ofhuman DU145 prostate tumor cells in nude mice in a xenograft study (FIG.6E).

Analysis and characterization of phospholipids and other complex lipidsrepresent another strategy to measure effects of small moleculeinhibitors on phospholipid metabolizing enzymes involved in tumorprogression, including but not limited to, LPAAT-β. Measurements ofphospholipids and other complex lipids may be derived from cell linescultured in vitro, from tissue or plasma in vivo (e.g., murine or otheranimal studies), or from human subjects (e.g., phlebotomy or biopsy).Phospholipids, which are the primary constituents of a cellular bilayer,contain a universal phosphoric acid residue connected to a glycerolbackbone. Phospholipid classes are defined by the chemical identity ofthe “head group” on the phosphoric acid moiety. However, eachphospholipid class is often a complex mixture of discrete molecularspecies due to the fact that the glycerol backbone has two substituentsresiding at the Sn1 and Sn2 position of attachment. The substituents areacyl chains and typically consist of long chain fatty acids but may alsoinclude a long chain ether, acetyl, or hydroxyl group. Chemicalmeasurements of phospholipids can involve a variety of analyticalmethods including, but not limited to, HPLC-MS (High Performance LiquidChromatography-Mass Spectrometry), HPLC-MS/MS (High Performance LiquidChromatography-Tandem Mass Spectrometry), one or two dimensional TLC(Thin Layer Chromatography), and radiometry. While all the statedmethods can be used to quantitate bulk mass changes in a particularphospholipid class of interest, mass spectrometry offers the uniqueability to measure all molecular species within a phospholipid class ina single measurement with a high degree of precision.

The above approach is demonstrated by performing HPLC-MS analyses ofphospholipid extracts from murine NIH/3T3 immortalized fibroblasts, bothnormal wild type, βHc2 cells (i.e., overexpressing LPAAT-β, and Hc2crecells (i.e., LPAAT-β gene removed by site-specific recombination).Analysis of phosphatidylinositol in these cell populations clearlyindicate a combined effect of LPAAT-β overexpression and cellulartransformation for the Hc2 population over that of the normal wild type.This effect is characterized by an increase in unsaturated (i.e.,palmitate and stearate) and monounsaturated (i.e., oleate) fatty acylchains indicated by an increased molecular abundance of ions at m/z 807,833, 835, 861, and 863 which correspond most likely tophosphatidylinositol species with acyl chains designated as 16:0-16:1,16:1-18:1 (and/or 16:0-18:2), 16:0-18:1, 18:1-18:1 (and/or 18:0-18:2),and 18:0-18:1, respectively. While multiple molecular species may resideat the same nominal mass, these species can be differentiated by tandem(MS/MS) mass spectrometry methods. Additionally, note that actualdetermination of positional location (i.e., Sn1 versus Sn2) requiresother analytical methods and only the most prevalent configuration islisted here. In addition to the increase in unsaturated andmonounsaturated acyl chains in the LPAAT-β overexpressing population(βHc2), there is also a corresponding decrease in polyunsaturated (i.e.,arachidonate) fatty acyl chains at m/z 857 (16:0-20:4) and m/z 885(18:0-20:4). Removal of the LPAAT-β transgene results inphosphatidylinositol distributions similar to that of the normal wildtype 3T3 cells.

In summary, endogenous LPAAT-β expression is detected at high levels byboth in situ hybridization and immunohistochemistry in particular tumortissues and often in surrounding stroma and is associated with tumorprogression. LPAAT-β overexpression appears to contribute reversibly totransformation and tumorigenesis of immortalized rodent cells and mayalso contribute to increased transformation of weakly tumorigenic humancell lines. Compounds selected from screening of LPAAT-β inhibitors fromdifferent structural families can inhibit proliferation of numeroustumor cell lines in vitro. Both nude and immunocompetent mice cantolerate at least 100 mg/kg/day for 4-5 days of the6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine maintaining bodyweight and overall health with no discernable gross pathology. Thiscompound can inhibit the growth of numerous tumor models in mice and maybe a tumor-static compound.

III. LPAAT-β Inhibitors.

In one aspect, the compounds of the present invention relate totriazines of the Formula:

wherein,

R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

R², R³, R⁴ and R⁵, each of which may be same or different, are hydrogen,alkyl, substituted alkyl, alkenyl, alkynyl, aryl or substituted aryl; orR² and R³ or R⁴ and R⁵, together with the nitrogen to which they areattached, form a piperidine, piperazine, or a morpholine ring; or

pharmaceutically acceptable salts thereof.

As used herein, “alkyl” refers to straight- or branched-chainhydrocarbons having from 1 to 10 carbon atoms and more preferably 1 to 8carbon atoms which includes, by way of example, methyl, ethyl, n-propyl,i-propyl, n-butyl, t-butyl and the like.

The term “alkyl” also refers to an “unsaturated alkyl” moiety, whichmeans that it contains at least one alkene or alkyne moiety. “Alkene” or“alkenyl” refers to a group consisting of at least two carbon atoms andat least one carbon-carbon double bond. “Alkyne” or “alkynyl” refers toa group consisting of at least two carbon atoms and at least onecarbon-carbon triple bond. The alkyl moiety, whether saturated orunsaturated, may be branched, non-branched, or cyclic.

“Substituted alkyl” refers to an alkyl group, preferably containing from1 to 10 carbon atoms, having from 1 to 5 substituents including halogen,hydroxyl, alkyl, aryl or substituted amino. A preferred substitutedalkyl group is trifluromethyl.

“Alkoxy” refers to the group “alkyl-O—” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy andthe like. “Substituted amino” refers to the group -NRR, wherein each Rgroup is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, or the Rgroups can be joined together with the nitrogen to form a heterocyclicring (e.g., piperidine, piperazine, or a morpholine ring).

“Aryl” refers to an unsaturated aromatic carbocyclic group of 6 to 14carbon atoms having a single ring (e.g., phenyl) or multiple condensedrings (e.g., naphthyl or anthryl).

“Substituted aryl” refers to aryl group which are substituted with 1 to3 substituents selected from hydroxy, alkyl, substituted alkyl, alkoxy,amino, aryl, —O—(CH₂)_(n)—O—(wherein n is an integer from 1 to 3),—(CH₂)_(m)—(wherein m is an integer from 3 to 5) or halogen.

“Cycloalkyl” refers to cyclic alkyl groups containing between 3 and 8carbon atoms having a single cyclic ring including, by way of example,cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.

“Halogen” or “halo” refers to fluoro, chloro, bromo, iodo. Mostpreferred halogens are chloro and fluoro.

“Mercapto” refers to the group -SR wherein the R group is independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, cycloalkyl, aryl or substituted aryl. The term “alkylmerecapto”refers to the group -SR when R is alkyl, substituted alkyl orcycloalkyl.

Compounds of the preferred embodiments of the present invention includethose compounds in Table 1. LPAATβ CT Colorimetric Assay NumberStructure IC₅₀ (nM) 31867

750 31942

400 31978

1,000 32028

200 32042

200 32099

650 116988

160 117147

3,800 31888

3,100

Preferred compounds include, but are not limited to,6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-cyclo-hexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine andN-(4-chloro-phenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.

Most preferred compounds include, but are not limited to,6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]-triazine-2,4-diamine.

The compounds of the preferred embodiments of the present inventioninhibit LPAAT-β and thereby inhibit cell proliferation. Therefore, thecompounds of the preferred embodiments of the present invention may beuseful in the treatment of cancer. The types of cancer that may betreated with the compounds of the preferred embodiments of the presentinvention include, but are not limited to, prostate, breast, lung,ovarian, brain, cervical, colon or bladder cancer, and not limited totumor cells expressing high levels of LPAAT-β as evidenced by thedecrease in NIH/3T3 Ki-ras tumor cell growth in vitro and in vivo whentreated with, 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine.

IV. Pharmacological Compositions, Therapeutic and Other Applications.

The compound of the present invention, or its pharmaceuticallyacceptable salt, can be administered to a human patient per se, or inpharmacological compositions where it is mixed with pharmaceuticallyacceptable carriers or excipient(s). Techniques for formulation andadministration of drugs may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

A. Routes of Administration.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intraperitoneal or intranasal injections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

B. Composition/Formulation.

Pharmacological compositions of the compounds and the pharmaceuticallyacceptable salts thereof are preferred embodiments of this invention.Pharmacological compositions of the present invention may bemanufactured by processes well known in the art; e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmacological compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated assterile aqueous solutions, preferably in physiologically compatiblebuffers such as Hanks' solution, Ringer's solution, or physiologicalsaline buffer. For transmucosal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmacological preparations fororal use can be made with the use of a solid excipient, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmacological compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration, e.g., bybolus injection or continuous infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmacological compositions for parenteral administration includesterile aqueous solutions of the active compounds in water soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation (see, for example, U.S. Pat.No. 5,702,717 for a biodegradable depot for the delivery of a drug).Such long acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Thepharmacological compositions herein also may comprise suitable solid orgel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

The compounds of the invention that inhibit LPAAT-β may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate,succinate, etc. formed by the reaction of an amino group with theappropriate acid.

C. Dosage.

Pharmacological compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount effective to achieve its intended purpose.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. For example, a dose can be formulated in animalmodels to achieve a circulating concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of LPAAT-β activity).Such information can be used to more accurately determine useful dosesin humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio betweenLD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (see e.g.,Fingl, et al., in “The Pharmacological Basis of Therapeutics,” (1975),Chapter 1, pp. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintainLPAAT-β inhibitory effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata; e.g., the concentration necessary to achieve 50-90% inhibition ofLPAAT-β using the assays described herein. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. However, HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner, of administration and the judgment of theprescribing physician. An exemplary systemic daily dosage is about 5 toabout 200 mg/kg of body weight. Normally, from about 10 to about 100mg/kg of body weight of the compounds of the preferred embodiments ofthe present invention, in one or more dosages per day, is effective toobtain the desired results. One of ordinary skill in the art candetermine the optimal dosages and concentrations of the compounds of thepreferred embodiments of the present invention with only routineexperimentation.

The compounds of the preferred embodiments of the present invention aresubstantially pure and preferably sterile. The phrase “substantiallypure” encompasses compounds created by chemical synthesis and/orcompounds substantially free of chemicals which may accompany thecompounds in the natural state, as evidenced by thin layerchromatography (TLC) or high performance liquid chromatography (HPLC).

D. Other Applications

The compounds of the preferred embodiments of the present invention maybe employed not only for therapeutic purposes, but also as aids inperforming research in vitro. For example, the compounds of thepreferred embodiments of the present invention may be used to studybiochemical pathways that would require the inhibition of LPAAT-β toelevated levels of LPA. Inhibition of LPAAT-β may result in theprolonged or limited activity of biochemical pathways that depend on, orrespond to, elevated levels of LPA.

Additionally, a cell culture medium comprising the compounds of thepreferred embodiments of the present invention is within the scope ofthe invention.

V. Assays for LPAAT-β DNA, RNA and Protein.

DNA molecules encoding the human LPAAT-β gene, or fragments thereof, canbe used to detect the level of LPAAT-β gene expression in tissuesamples. Such a detection method can be used, for example, to comparethe amount of LPAAT-β RNA in a sample obtained from normal tissue and ina sample isolated from methotrexate-resistant tumor tissue. The presenceof relatively low levels of LPAAT-β RNA in the tumor sample wouldindicate that methotrexate resistance is due, at least in part, tounderexpression of the LPAAT-β gene.

In testing a tissue sample for LPAAT-β RNA using a nucleic acidhybridization assay, RNA can be isolated from tissue by sectioning on acryostat and lysing the sections with a detergent such as SDS and achelating agent such as EDTA, optionally with overnight digestion withproteinase K. Such tissue may be obtained by biopsy. A preferredquantity of tissue is in the range of 10-100 milligrams. Protein may beremoved by phenol and chloroform extractions, and nucleic acids areprecipitated with ethanol. RNA may be isolated by chromatography on anoligo dT column and then eluted from the column. Further fractionationcan also be carried out according to methods well known to those ofordinary skill in the art.

A number of techniques for molecular hybridization are used for thedetection of DNA or RNA sequences in tissues. When large amounts oftissue are available, analysis of hybridization kinetics provides theopportunity to accurately quantitate the amount of DNA or RNA present,as well as to distinguish sequences that are closely related but notidentical to the probe. Reactions are run under conditions ofhybridization (T_(m)−25° C.) in which the rate of re-association of theprobe is optimal. Wetmur et al., J. Mol. Biol. 31:349 (1968). Thekinetics of the reaction are second order when the sequences in thetissue are identical to those of the probe; however, the reactionexhibits complex kinetics when probe sequences have partial homology tothose in the tissue. Sharp et al., J. Mol. Biol. 86:709 (1974).

The concentration of probe to cellular RNA is determined by thesensitivity desired. To detect one transcript per cell would requireabout 100 pg of probe per mg of total cellular DNA or RNA. The nucleicacids are mixed, denatured, brought to the appropriate saltconcentration and temperature, and allowed to hybridize for variousperiods of time. The rate of reassociation can be determined byquantitating the amount of probe hybridized either by hydroxyapatitechromatography (Britten et al., Science 161:529 (1968)) or by S1nuclease digestion (Sutton, Biochim. Biophys. Acta 240:522 (1971)).

Another method of hybridization is the Northern Blot technique. Theparticular hybridization technique is not essential to the invention,and any technique commonly used in the art is within the scope of thepresent invention. Typical probe technology is described in U. S. Pat.No. 4,358,535, incorporated by reference herein. For example,hybridization can be carried out in a solution containing 6×SSC (10×SSC:1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0), 5× Denhardt's (1×Denhardt's: 0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.02%Ficoll 400), 10 mM EDTA, 0.5% SDS and about 10⁷ cpm of nick-translatedDNA for 16 hours at 65° C.

The aforementioned hybridization assays are particularly well suited forpreparation and commercialization in kit form, the kit comprising acarrier means compartmentalized to receive one or more container means(vial, test tube, etc.) in close confinement, with each container meanscomprising one of the separate elements to be used in hybridizationassay. For example, there may be a container means containing LPAAT-βDNA molecules suitable for labeling by “nick translation,” or containinglabeled LPAAT-β DNA or labeled LPAAT-β RNA molecules. Further containermeans may contain standard solutions for nick translation of DNAcomprising DNA polymerase I/DNase I and unlabeled deoxyribonucleotides.

Antibodies to human LPAAT-β protein can be obtained using the product ofan LPAAT-β expression vector as an antigen. The preparation ofpolyclonal antibodies is well-known to those of skill in the art. See,for example, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), pp. 1-5 (Humana Press 1992).Alternatively, an LPAAT-β antibody of the present invention may bederived from a rodent monoclonal antibody (MAb). Rodent monoclonalantibodies to specific antigens may be obtained by methods known tothose skilled in the art. See, for example, Kohler and Milstein, Nature256:495, 1975, and Coligan et al. (eds.), Current Protocols inImmunology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter“Coligan”]. Briefly, monoclonal antibodies can be obtained by injectingmice with a composition comprising an antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof techniques that are well known in the art. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, 10:79 (Humana Press, Inc. 1992). A LPAAT-β antibody may also bederived from a subhuman primate antibody. General techniques for raisingtherapeutically useful antibodies in baboons may be found, for example,in Goldenberg et al., International Patent Publication No. WO 91/11465(1991), and in Losman et al., Int. J Cancer 46:310 (1990).

Alternatively, a therapeutically useful LPAAT-β antibody may be derivedfrom a “humanized” monoclonal antibody. Humanized monoclonal antibodiesare produced by transferring mouse complementary determining regionsfrom heavy and light variable chains of the mouse immunoglobulin into ahuman variable domain, and then, substituting human residues in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by the publication of Orlandi etal., Proc. Nat'l. Acad. Sci. USA 86:3833 (1989). Techniques forproducing humanized MAbs are described, for example, by Jones et al.,Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988);Verhoeyen et al., Science 239:1534 (1988); Carter et al., Proc. Nat'l.Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437(1992); and Singer et al., J. Immun. 150:2844 (1993), each of which ishereby incorporated by reference.

As an alternative, a LPAAT-β antibody of the present invention may bederived from human antibody fragments isolated from a combinatorialimmunoglobulin library. See, for example, Barbas et al., METHODS: ACompanion to Methods in Enzymology 2:119 (1991); and Winter et al., Ann.Rev. Immunol. 12:433 (1994) which are incorporated herein by reference.Cloning and expression vectors that are useful for producing a humanimmunoglobulin phage library can be obtained, for example, fromSTRATAGENE Cloning Systems (La Jolla, Calif.). In addition, a LPAAT-βantibody of the present invention may be derived from a human monoclonalantibody. Such antibodies are obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856(1994); and Taylor et al., Int. Immun. 6:579 (1994).

Having now generally described this invention, the same will beunderstood by reference to the following examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE 1 PRODUCTION OF RECOMBINANT LPAAT-β FOR VARIOUS ASSAYS

For the construction of Baculovirus expression vectors, the full-lengthhuman LPAAT-β cDNA was amplified by PCR from the DNA templatepCE9.LPAAT-β (West et al., DNA Cell Biol. 16:691-701 (1997)) using theprimers 5′- TGATATCCGA AGAAGATCTT ATGGAGCTGT GGCCGTGTC-3′ (olpb1F; SEQID NO:3) and 5′-CAGGCTCTAG ACTACTGGGC CGGCTGCAC-3′ (olpb1R; SEQ IDNO:4). The ˜870 bp fragment generated was reamplified by PCR using theprimers 5° CCTACGTCG ACATGGAACA AAAATTGATA TCCGAAGAAG ATC-3′ (olpb2F;SEQ ID NO:5) and 5′-CAGGCTCTAG ACTACTGGGC CGGCTGCAC-3′ (olpb1R; SEQ IDNO:6). The ˜890 bp fragment generated was then cleaved with Sal I andXba I for insertion into pFastBac™ HTc vector (Life Technologies,Gaithersberg, Md.) between the Sal I and Xba I sites for the generationof the plasmid pFB.LPAAT-β. This plasmid was then transformed into E.coli DH1OBac™ (Life Technologies, Gaithersberg, Md.) for the generationof recombinant Bacmid DNA for transfection into HighFive (Invitrogen,San Diego, Calif.) or SF9 insect cells for the production of recombinantBaculovirus stocks using the protocol described in the Bac-to-Bac®Baculovirus Expression System (Life Technologies, Gaithersberg, Md.), aeukaryotic expression system for generating recombinant baculovirusthrough site- specific transposition in E. coli. Viral stocks harvestedfrom the transfected cells can then be used to infect fresh insect cellsfor the subsequent expression of LPAAT-β fusion protein with apoly-histidine tag and a myc-epitope near its N-terminus. The membranefraction from these Sf9 cells would be the source of LPAAT enzyme.

EXAMPLE 2 PREPARATION OF CELL MEMBRANES FROM SF9 CELLS

For the preparation of membranes From Sf9 Cells, all steps are performedon ice or at 4° C. Sf9 cell pellets (˜10⁸ cells) were thawed andresuspended in 1-2 ml of buffer A (20 mM Hepes, pH 7.5, 1 mM DTT, 1 mMEDTA, 20% w/v glycerol, 1 mM Benzamidine, 1 μg/ml soybean trypsininhibitor (SBTI), 1 μg/ml pepstatin A) w/o DTT but with 1 mM Pefabloc.The cells were lysed by sonication using a Branson Sonifier at output=2, duty cycle=2, 10 pulses each at 10 s. with the tip of smallsonicator probe submerged but not touching the walls. DTT was then addedto 1 mM from a 1 M stock. The samples were centrifuged at 1500 rpm for 5min. The low speed supernatant was saved and centrifuged (TLA 100.3rotor, polycarbonate tubes, 2 ml/tube or 1.5 ml/tube minimum) at100000×g for 1 hr. The high speed pellet was resuspend in Buffer A witha probe sonicator (10 pulses @ output #2 and duty cycle 20%) as a sourceof LPAAT enzyme.

EXAMPLE 3 ASSAY OF LPAAT-p ACTIVITY

LPAAT-β catalyzes the transfer of an acyl group from a donor such asacyl-CoA to LPA. The transfer of the acyl group from acyl-CoA to LPAleads to the release of free CoA, which can be reacted with the thiolreagent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). The reactionbetween DTNB and the free sulfhydryl group from CoA generates ayellow-colored product, 3-carboxylato-4-nitrothiophenolate (CNP), thatabsorbs at 413 nm. LPAAT-β derived from Sf9 cell membrane overexpressingLPAAT-β were resuspended in HEPES saline buffer (20 mM HEPES pH 7.5, 150mM NaCl), 1 mg/ml BSA and 72 μl aliquots were distributed into 96-wellmicrotiter plates. 8 μl of compound of interest at 200 μM dissolved in100% DMSO was added into each well. 20 μl of 1 mM 18:1-CoA and 1 mMsn−1-18: 1 lysoPA was then added to each well to initiate the reactionand allowed to run at room temperature for 25 min. 100 μl of I mM DTNBin 100% ethanol was then added to each well to quench the reaction andfor color development. The absorbance at 405 nm, measured using aspectrophotometer plate reader, is proportional to the activity ofLPAAT-β in the sample. This colorimetric assay was used for the highthroughput screening of LPAAT inhibitors. Compounds that showed >50%inhibition of the change in absorbance at 405 nm compared to controlwere selected for a secondary assay. FIG. 7 shows an example of thecalorimetric assay of which the time course of color development isdependent on the amount of LPAAT enzyme added. FIG. 8 shows an exampleof the results obtained from assaying a plate of various compounds at 16μM. Compounds that gave a reading of less than 0.06 arbitrary units(indicated by arrow on right margin) were selected for further study.

A secondary assay for LPAAT activity in cell extracts based on eitherthe conversion of fluorescent NBD-LPA to NBD-PA (West, et al., DNA CellBiol. 6: 691-701, 1997) or [¹⁴C]LPA to [¹⁴C]PA using TLC analysis wasused to screen compounds that showed >50% inhibition of LPAAT activityin the primary calorimetric assay. The radiometric assay was carried outin Sf9 cell membrane overexpressing LPAAT-β resuspended in HEPES-salinebuffer, pH 7.5, 1 mg/ml BSA, 1 mM EDTA and 200 μM [¹⁴C]18:1-CoA and 200μM sn−1-18:1 lysoPA. The samples were incubated 7 min at 37° C.extracted into organic solvent (CHCl₃/CH₃OH/HCl at 33/66/1), beforeloading onto TLC plates. A more detailed protocol for the radiometricassay is described below:

Specifically, this LPAAT assay is a modification of the acyltransferaseassay published previously (Hollenback and Glomset, Biochemistry 37:363-376 (1999)).

1. The basic assay, in a total vol of 50 μl, employs a solution ofsubstrates and the protein sample. Total assay volume, as well as thevolume of each solution, can be changed to fit an experiment. Inaddition, other compounds, ex inhibitors and activators, can be includedin the assay as well.

2. To prepare the solution of substrates:

-   -   a. Stocks of Hepes (pH 7.5), NaCl, EDTA, BSA and acyl-CoA (from        Serdery or Sigma) are mixed with water to make the appropriate        concentration of each compound. This can be varied from        assay-to-assay, but the final reaction mix is about 50 mM Hepes,        100 mM NaCl, 1 mM EDTA, 1 mg/ml BSA and 0-400 μM acyl-CoA.    -   b. The lysoPA (from Avanti) is typically stored in chloroform        and the ¹⁴C-labeled acyl-CoA (from Amersham) is typically stored        in water/ethanol=1:1. Appropriate amounts of each solution are        added the to a 12×75 mm borosilicate glass test tube and dry the        solvent under N₂ or Ar. An appropriate volume of the solution        prepared in 2 a is added to the iysoPA and ¹⁴C-labeled acyl-CoA.        The lipids are resuspend by sonication for 15 sec in a bath        sonicator. The resulting suspension is then incubated (with        occasional gentle vortexing) for about 10 minutes at room temp.        The sn−1-16:0 lysoPA may require brief warming of the solvent to        solubilize it. The concentration of lysoPA and ¹⁴C-labeled        acyl-CoA can vary, but typically the final lysoPA concentration        ranges between 0 and 400 μM and the ¹⁴C-labeled acyl-CoA        specific activity ranges between 0.5 and 2 Ci/mol.

3. Protein sample: varies from experiment-to-experiment.

4. The assay is performed by mixing the components in 12×75 mmborosilicate glass test tubes (the order of addition does not matterunless indicated) and incubating at 37° C. for 5 to 10 minutes such thatthe assay within the linear range for time and protein.

5. The reaction is quenched by adding 1.3 ml ofchloroform/methanol/HCl=48/51/0.7 and vortexing. 10 μl of carriersolution is then added (3 mg/ml each PA, ex. 16:0-18:1, and lysoPA, exsn−1-18:1, in chloroform). Two phases are formed by adding 0.3 ml ofwater to each tube and vortexing.

6. The sample is centrifuged for 3 minutes at 1000×g, the upper(aqueous/methanol) phase is aspirated and the lower phase is dried undernitrogen.

7. Thin layer chromatography:

-   -   a. The dried samples are resuspended in 50 pl of chloroform and        a 15 μl aliquot is immediately spotted on an Analtech silica gel        60 HP-TLC plate (10×20 cm).    -   b. Plates are developed in chloroform/methanol/acetic        acid/water=85/12.5/12.5/3 (takes about 15 min) and dried.    -   c. To be able to convert pixel volume (determined by the Storm        phosphor imager, see step 8b) into cpm, cpm standard curve must        be generated on the plate. ¹⁴C-labeled oleate dilutions in        chloroform are made for this purpose. Four stocks (50 cpm/μl to        800 cpm/μl) are made and 2 μl of a different concentration are        spotted in each corner of the plate (where previously there was        no radioactivity).    -   d. For quality control purposes, the plates are stained with        primuline and scanned with the Storm (blue chemilluminescence        mode).

The PA and lysoPA bands are easily detected in this system because ofthe carrier added in step 5. PA and lysoPA have respective Rf's of about0.63 and 0.21.

8. Quantitating activity:

-   -   a. The plates are then wrapped in saran wrap and exposed to a        freshly blanked phosphor screen overnight (longer exposures can        also be done to increase the signal).    -   b. The screens are scanned (Phosphorimager mode), and LPAAT        activity is determined by quantifying the pixels in the band        comigrating with PA standard versus the standard curve generated        from the cpm standards that were spotted in step 7c.

FIG. 9 shows examples of a compound, namely,6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine, selected from thesecondary screening that exhibit concentration-dependent inhibition ofLPAAT-β activity (∘). Moreover, these compounds have minimal effect onLPAAT-α activity (□), suggesting they are isoform-specific inhibitors.

EXAMPLE 4

Triazines of the preferred embodiments of the present invention weresynthesized by one of two methods. Symmetrically substituted triazineswere synthesized by the addition of two equivalents of the appropriateamino compound, in the presence of diisopropylethylamine, to cyanuricchloride. Non-symmetrical triazines were synthesized in a stepwisefashion by the sequential addition of the amino compound in the presenceof potassium carbonate with isolation of the intermediatemono-amino-dichlorotriazine.

A. Method 1: Synthesis of Symmetrical Triazines.

SYNTHESIS OF 6-CHLORO-N,N′-DIPHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-113020)

To a mixture of cyanuric chloride (5.07 g, 27.5 mmol) and acetonitrile(60 ml), cooled in an ice bath, was added a solution of aniline (5.3 ml,58.2 mmol) and diisopropylethylamine (10.5 ml, 60.3 mmol) inacetonitrile (15 ml) over 30 minutes. After stirring at room temperaturefor 20 hours, the mixture was filtered. The solid was washed with ethylacetate (4×25 ml), suspended in water (50 ml), stirred for 1 hour,filtered, washed with water, and dried under vacuum to give6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine (3.97 g, 48% yield)as a white solid. ¹³H NMR (d₆-DMSO)δ 7.61 (2H, br s), 7.28-7.37 (4H, m),7.05-7.11(4H, m). ¹³C NMR (d₆-DMSO) δ 167.7, 164.0, 138.5, 128.4 124.5,121.3.

B. Method 2: Synthesis of Unsymmetrical Triazines.

SYNTHESIS OF6-CHLORO-N-(4-CHLOROPHENYL)-N′-PHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-116433)

To a mixture of cyanuric chloride (5.15 g, 27.9 mmol), potassiumcarbonate (3.98 g, 28.8 mmol) and 18-crown-6 (158 mg, 0.60 mmol) intoluene (40 ml), cooled in an ice bath, was added a solution of4-chloroaniline (3.61 g, 28.3 mmol) in toluene (20 ml) over 15 minutes.After stirring at room temperature for 20 hours, the mixture was treatedwith ethyl acetate (60 ml) and filtered through a pad of celite undersuction. The filtrate was concentrated under vacuum and recrystallized(chloroform) to give(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (4.06 g, 53%yield) as a white solid. ¹H NMR (d₆-DMSO) δ 11.23 (1H, s), 7.62 (2H, d,J=9 Hz), 7.47(2H, d, J=9 Hz). ¹³C NMR (d₆-DMSO) δ 169.5, 168.5, 163.6,135.7, 128.6, 122.5, ESMS m/z 273 (M−H)⁻.

To a mixture of(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (3.97 g, 14.4mmol), potassium carbonate (2.20 g, 15.9 mmol) and 18-crown-6 (46 mg,0.17 mmol) in toluene (25 ml), cooled in an ice bath, was added asolution of aniline (1.4 ml, 15.4 mmol) in toluene (10 ml) over 15minutes. After stirring at room temperature for 24 hours, the mixturewas treated with ethyl acetate (35 ml) and filtered through a pad ofcelite under suction. The filtrate was concentrated under vacuum and theresidue was recrystallized (chloroform) to give6-Chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine (2.20g, 46% yield) as a white solid. ¹H NMR (d₆-DMSO) δ 10.1-10.4 (2H, br s),7.5-7.8 (4H, br s), 7.3-7.5 (4H, m), 7.15-7.05 (1H, m), ESMS m/z 330(M−H)⁻.

EXAMPLE 5 SYNTHESIS OF6-CHLORO-N-(4-CHLOROPHENYL)-N′-(4-METHOXY-PHENYL)-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-31867)

The reaction of(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine withp-anisidine, according to method 2, gave6-chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamine(51 mg, 62%): ¹H NMR (CDCl₃) 7.21-7.50 (6 H, m), 6.91(2H, d, J=11 Hz),3.85 (3H, s).

EXAMPLE 6 SYNTHESIS OF6-CHLORO-N-(4-METHOXYPHENYL)-N′-PHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-31942)

To a solution of 2-(4-methoxyphenyl)amino-4,6-dichloro-1,3,5-triazine(57 mg, 0.21 mmoles) in acetonitrile (0.5 ml) was added a solution ofaniline (0.021 ml, 0.23 mmoles) and diisopropylethylamine (0.05 ml, 0.29mmoles) in acetonitrile (0.5 ml). After stirring for 24 hours, themixture was concentrated under vacuum and the residue was dissolved inethyl acetate (10 ml). The solution was washed with a solution composedof saturated aqueous sodium chloride solution and 1 M hydrochloric acid(1:1, 2×10 ml), dried over sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified by flash chromatography on silicagel eluting with 10% ethyl acetate-hexane to provide CT-31942 (38 mg,57% yield). ¹H NMR (d₆-DMSO) δ 10.00-10.22 (m, 2H), 7.26-7.82 (m, 6H),7.01-7.10 (m, 1H), 6.91 (d, J=9 Hz, 2H), 3.74 (s, 3H).

EXAMPLE 7 SYNTHESIS OFN-BENZO[1,3]DIOXOL-5-YL-6-CHLORO-N′-(4-CHLOROPHENYL)-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-31978)

The reaction of 4,6-dichloro-N-(4-chlorophenyl)-[1,3,5]triazine-2-aminewith 3,4-methylenedioxyaniline using the method described for thesynthesis of CT-116433 provided CT-31978 (56 mg, 65% yield). ¹H NMR(d₆-DMSO) δ 10.08-10.37 (m, 2 H), 7.58-7.87 (m, 2H), 7.21-7.40 (m, 3H),6.85-7.03 (m, 2H), 6.00 (s, 2H).

EXAMPLE 8 SYNTHESIS OF6-CHLORO-N-(2,3-DIHYDROBENZO[1,4]DIOXIN-6-YL)-N′-PHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-32028)

To a solution of 4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine (95 mg,0.39 mmoles) in tetrahydrofuran (2 ml) was added a solution of1,4-benzodioxan-6-amine (64 mg, 0.42 mmoles) and triethylamine (0.07 ml,0.50 mmoles) in tetrahydrofuran (0.5 ml). After stirring for 24 hours,the mixture was concentrated under vacuum and the residue was dissolvedin ethyl acetate (10 ml). The solution was washed with a solutioncomposed of saturated aqueous sodium chloride solution and 1 Mhydrochloric acid (1:1, 2×10 ml), dried over sodium sulfate, filtered,and concentrated under vacuum. The residue was purified by flashchromatography on silica gel eluting with 25% ethyl acetate-hexanes toprovide CT-32028 (97 mg, 69% yield). ¹H NMR (d₆-DMSO) δ 9.88-10.28 (m,2H), 7.60-7.85 (m, 2H), 7.20-7.40 (m, 3H), 6.96-7.10 (m, 2H), 6.77-6.81(m, 1H), 4.21 (s, 4H).

EXAMPLE 9 SYNTHESIS OFN-BENZO[1,3]DIOXOL-5-YL-6-CHLORO-N′-PHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-32042)

The reaction of 4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine with3,4-methylenedioxyaniline using the method described for the synthesisof CT-32028 provided CT-32042 (51 mg, 42% yield). ¹H NMR (d₆-DMSO) δ10.05-10.29 (m, 2H), 7.57-7.80 (m, 2H), 7.21-7.42 (m, 3H), 6.80-7.13 (m,3H), 6.03 (s, 2H).

EXAMPLE 10 SYNTHESIS OF6-CHLORO-N-INDAN-5-YL-N′-PHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE (CT-32099)

The reaction of 4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine with5-aminoindan using the method described for the synthesis of CT-32028provided CT-32099 (36 mg, 37% yield). ¹H NMR (d₆-DMSO) δ 10.15-10.28 (m,2H), 7.56-7.72 (m, 3H), 7.05-7.39 (m, 5H), 2.75-90 (m, 4H), 1.94-2.09(m, 2H).

EXAMPLE 11 SYNTHESIS OF6-CHLORO-N-2-(4-CHLOROPHENYL)-N-4-PROPYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-116988)

The reaction of 4,6-dichloro-N-(4-chlorophenyl)-[1,3,5]triazine-2-aminewith propylamine using the method described for the synthesis ofCT-116433 provides CT-116988.

EXAMPLE 12 SYNTHESIS OFN-(4-CHLOROPHENYL)-6-METHOXY-N′-PROPYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-117147)

A mixture of CT-116988 and sodium methoxide (3.0 molar equivalents) inmethanol is heated at reflux for 18 hours. After cooling to roomtemperature, the reaction mixture is concentrated under vacuum. Theresidue is suspended in ethyl acetate and washed with water. The ethylacetate phase is dried over sodium sulfate, filtered and concentratedunder vacuum. The residue is purified by flash chromatography on silicagel to provide CT-117147.

EXAMPLE 13 SYNTHESIS OFN-(4-CHLOROPHENYL)-6-METHYLSULFANYL-N′-PHENYL-[1,3,5]TRIAZINE-2,4-DIAMINE(CT-31888)

A mixture of CT-116433 (108 mg, 0.32 mmol) and sodium methanethiolate(79 mg, 1.13 mmol) in dimethyl sulfoxide (3 ml) was heated at 70° C. for18 hours. After cooling to room temperature the mixture was diluted withethyl acetate (25 ml) and was washed with a 1:1 solution of water andsaturated aqueous sodium chloride solution (4×25 ml). The organic phasewas dried over sodium sulfate, filtered, and concentrated under vacuum.The residue was purified by flash chromatography on silica gel elutingwith 5% ethyl acetate-hexane to provide CT-31888 (76 mg, 69% yield). ¹HNMR (d₆-DMSO) δ 7.51-7.60 (m, 4H), 7.26-7.39 (m, 5H), 7.00-7.17 (m, 2H),2.56 (s, 3H).

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

1. A compound selected fromN-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,6-chloro-N-cyclo-hexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chlorophenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamineand 6-chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]-triazine-2,4-diamine,or pharmaceutically acceptable salts thereof.
 2. A compound of claim 1,wherein the compound isN-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine.
 3. Acompound of claim 1, wherein the compound isN-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine.
 4. Acompound of claim 1, wherein the compound is(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine.5. A compound of claim 1, wherein the compound is6-chloro-N-cyclo-hexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine.
 6. Acompound of claim 1, wherein the compound is2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol.7. A compound of claim 1, wherein the compound is6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.
 8. Acompound of claim 1, wherein the compound is6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine.9. A compound of claim 1, wherein the compound isN-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine.
 10. Acompound of claim 1, wherein the compound is2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol.
 11. Acompound of claim 1, wherein the compound isN-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine.
 12. Acompound of claim 1, wherein the compound is6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine. 13.A compound of claim 1, wherein the compound isN-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine.14. A compound of claim 1, wherein the compound is6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine.15. A compound of claim 1, wherein the compoundN-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine.16. A compound of claim 1, wherein the compound is6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine.
 17. Acompound of claim 1, wherein the compound is6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine. 18.A compound of claim 1, wherein the compound isN-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine. 19.A compound of claim 1, wherein the compound is6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine.20. A compound of claim 1, wherein the compound is6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine.
 21. Acompound of claim 1, wherein the compound isN-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine.
 22. Acompound of claim 1, wherein the compound is6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine.
 23. A compound ofclaim 1, wherein the compound isN-tert-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine.24. A compound of claim 1, wherein the compound is6-chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamine.25. A compound of claim 1, wherein the compound is6-chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]-triazine-2,4-diamine. 26.A pharmaceutical composition comprising the compound of any one ofclaims 1-25 and a pharmaceutically acceptable carrier.